Immunomodulation against microbial pathogens through dendritic cells

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________

Immunomodulation against microbial pathogens through dendritic cells J. M. Silva1,2, E. Zupancic1, C. Peres1, L.C. Silva1, R. Gaspar1, V. Préat2 and H. F. Florindo1,* 1

iMed.UL – Research Institute for Medicines and Pharmaceutical Sciences; Nanomedicines & Drug Delivery Systems Group, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, Edf. E, 1649-003 Lisbon, Portugal; 2 Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Université Catholique de Louvain, Avenue Mounier, B1 73.12, 1200 Brussels, Belgium. Dendritic cells (DCs) are the most potent antigen presenting cells (APCs), being a key player in the regulation of the adaptive immune response. These immune cells are in their immature form in circulation and peripheral tissues, being able to take up, process and activate the antigen-presenting pathway by the interaction of antigen-loaded Major Histocompatibility Complex molecules class I (MHC class I) and II with, respectively,CD4+ and CD8+ T cells, which will then stimulate Cytotoxic T Lymphocytes (CTL) and Th cells. As a result, this heterogeneous population, present in lymphoid and nonlymphoid organs, is fundamental to survey tissues for foreign antigens and thus to induce an effective immune response, but also, on the other hand, to maintain tolerance against self or harmless antigens. In this review we will explore the interplay between DCs subpopulations and microbial pathogens, highlighting their role as regulators in health and infectious diseases. In addition, we will emphasize progress in our understanding of immunomodulatory strategies, namely those based on nanodelivery systems used to promote host protection through the delivery of antigens to DCs and their presentation to T-cells, being these important factors to have in consideration when developing an effective vaccine. Keywords Dendritic cells, immune system, host-pathogen interactions, immunomodulation, nanoparticles

1. General remarks The infection pathogenesis is an overall result of a continuous battle between host and microbial pathogens. Over the last decade, new insights were brought into the characterisation of host immune response to bacterial pathogens, which has been underlying the development of new strategies to modulate immune system against those infecting microbes. Within those cellular events, specific mechanisms that mediate the membrane trafficking and intracellular pathogen elimination have been elucidated, contributing for a better characterization of innate immune mechanisms, which clearly depends on the effective interaction of multiple signalling pathways [1]. One of those innate immune pathways involves the recognition of pathogen-associated molecular patterns (PAMPs) to detect infectious agents, which work in parallel with several other protection mechanisms, depending on the subcellular compartment occupied by a specific pathogen [2, 3]. In addition, the increasing knowledge regarding specific mechanisms used by pathogens to evade hostcell innate responses has resulted in a better understanding of the host protective pathways. The central role of DCs regarding the activation and regulation of innate and adaptive immune responses has lead to the evolution of strategies used by microbial pathogens to prevent their recognition by these potent phagocytic cells, and further bacteria-specific T cell activation [3]. Among those strategies, it is possible to emphasize the manipulation of host immune system through the inhibition of antigen-MHC complex presentation to T cells or even modulation of the important Th1/Th2 balance of T cells [2]. Antigen recognition and DCs migration are highly controlled and involve several mediators and their receptors. The interaction among B cells, T cells and mature DCs results in broaden and integrated immune response, also fundamental for the establishment of an immunological memory [4, 5]. As a result, any impairment of DCs function will be vital for bacterial survival and dissemination inside host. Several approaches have been explored in order to improve immune responses against infectious agents [6,7]. Particulate adjuvants, such as poly(lactic-co-glycolic acid) (PLGA) and poly-ε-caprolactone (PCL) nanoparticles (NPs), have generated a lot of interest due to their biodegradability, biocompatibility and mechanical strength [7]. These NPs can also act as adjuvants, maintaining the antigenicity and immunogenicity of encapsulated biomolecules. PLGA, used for decades in humans, is the most studied polymer for vaccine formulation and has been able to increase humoral and cellular responses to antigen-loaded PLGA NPs. PCL has also a great potential for developing antigen controlled release matrices by its low degradation rate, hydrophobicity, good drug permeability, in vitro stability and low toxicity [7]. Polymeric NP-based strategies used to modulate host immune system, mainly through DCs activation/migration will be discussed, as well as, the regulatory issues underlying their approval by authorities.

2. Host immune defence mechanisms and bacterial pathogens evasion strategies 2.1. Main immune defence mechanisms to bacterial pathogens The human host, as a multicellular eukaryotic organism, has diverse, complex and extremely coordinated antimicrobial recognition and non-specific (e.g. mechanical barriers, secretions, commensal flora and mucus) or specific defence

1686

© FORMATEX 2013


systems that control the interaction between host-microbial agents, dictating the overall outcome. The microbial agent, non-pathogenic or pathogenic, can be categorized in extracellular, intracellular or encapsulated bacteria, and will be responsible for the induction of different patterns of the immune responses. The outcome will depend on virulence factors that dictate the microbial ability to evade host barriers and overcome the commonly triggered immune response [1]. The mucosal epithelial surfaces (mouth, eye, nose, rectum and vagina), constitute the most common portal of entry for microbial pathogens, being the first line of host defence against further infections [8-10]. Depending on the epithelial sites, different mucosal cells are vital for physical barriers such as: 1) goblet cells able to produce mucus that will form a dense overlay covering the entire epithelium, and trefoil peptides important for epithelial growth and repair; 2) enteroendocrine cells that have a paracrine effect due to neuroendocrine molecules production; 3) paneth cells at gastrointestinal tract, responsible for the production of anti-microbial peptides or defensins; 4) ciliated cells at the upper respiratory tract and 5) the peristalsis initiated by the brush border of gut epithelial cells [10-12]. The mucus constitutes a thick and enzymatic barrier, which rheological properties result from the non-covalent bond formed between mucin glycoproteins of high molecular weight that also confers its negative charge at neutral pH [13, 14]. The viscosity and thickness of this layer, along with the mucociliaty clearance and ciliary beating at nasal mucosa, will prevent microbial adherence and interpenetration, contributing for host protection from environmental pathogens. The interaction of microbes with these barriers triggers a multistep and coordinated process that begins with the innate immune recognition of the microbial pathogen, constituting the critical step in the early control of bacterial replication and successful eradication of an infection. The innate immune system, in turn, alerts the body to an infection and ultimately activate the adaptive immune mechanisms mediated by B and T cells, which helps clear the infection and builds specific immunity with a memory component [3, 11, 15]. In addition, molecules as defensins, α-defensins in Paneth cells’apical granules and β-defensins founded in gingival, gastrointestinal and airway mucosa, are antimicrobial entities from the innate immune system able to actively interact with adaptive immunity, as they present a chemotactic activity directed to DCs and T cells [11]. The common mucosal immune system (CMIS) is an interconnected structure present in mammals that highly contributes for the protection of those vast epithelial surface areas due to organized lymphoid tissues, being thus critical for the induction of effective mucosal immune responses [10, 12]. The CMIS comprises inductive and effector sites connected via the lymphatic system, which constitutes the basis of cellular immune response at those mucosal surfaces. The epithelial barriers at different mucosal sites present a mucosaassociated lymphoid tissue (MALT) with different structures according to the body region. This network includes mucosal inductive sites as gut-associated lymphoid tissues (GALT), nasopharyngeal-associated lymphoid tissues (NALT), bronchial-associated lymphoid tissue (BALT) that contains B cell regions, T cell regions and APCs responsible for the induction of a pathogen-specific immune response. GALT is the largest lymphatic organ where a large amount of plasma cells produce high levels of IgA, the predominant immunoglobin class all over the body organism [11]. These organized lymphoid tissues are covered by follicle-associated epithelium (FAE) characterized by a special type of membranous epithelial cells, called microfold cells (M cells). These cells possess a disorganized and sparse brush border at their apical site that allows the uptake of antigens at the mucosa lumen, and further transport to follicular environment (Peyer’s patches), where DCs will capture and process those antigens, initiating a mucosal immunity by their delivery to immunologically active cells (B and T cells, neutrophils, mast cells, eosinophils and macrophages) [12, 16]. Mucosal epithelial cells are able to produce immunologically active proteins, as cytokines, chemokines, in response to proinflammatory cytokines. Lymphocyte are key components of this CMIS:1) intraepithelial lymphocytes, mostly CD8+ T cells present at the basolateral side of enterocytes, containing CD45RO+, perforin and adhesive molecules (integrin αEβ7); 2) lymphocytes diffused at lamina propria, being mainly represented through CD4+ T cells and B cells responsible for the production of polymeric immunoglobulins, mainly secretory immunmoglobulins of the IgA isotype [10, 11]. IgA is a vital humoral defence factor for protective immune responses at mucosal surfaces. IgA polymeric form is predominant at secretions, while its monomeric form is mostly founded in circulation. This secretory IgA is thus a dimeric molecule and its protective functions result from a molecular structure that allows its penetration into secretions, resistance to enzymatic activities and proteolysis, and effector function at mucosal surfaces, preventing bacteria adherence to host mucosal surfaces and antigen penetration and dissemination through the organism. In addition, this immunoglobulin is able to transport bactericidal molecules, such as lactoferrin and lactoperoxidase, to bacterial surfaces [3, 11]. Therefore, CMIS constitutes a multiphasic process resultant from the migration of lymphocytes from inductive parts to effector sites, extremely important for the immune events triggered by a microbial invasion. 2.2. Immune evasion by microbial pathogen Despite this complex and extremely effective mucosal network, the pressure of survival has engendered a fascinating range of adaptations in organisms. Higher organisms have developed very sophisticated defence mechanisms that are designed to control the resident microflora and to prevent the continual microbial insult of the host’s environment. In

© FORMATEX 2013

1687


fact, microbial pathogens have coevolved with their hosts and thereby developed highly complex and efficient strategies to overcome both innate and adaptive immune mechanisms, which play a critical role in their ability to cause disease or chronic infection. Mycobacterium tuberculosis and Helicobacter pylori are examples of microbial agents that are able to coevolved with their host, establishing an equilibrium that causes persistent but minor pathologies [1, 3, 17]. Among all strategies used by microbial organisms to overcome the immune response, it is possible to emphasize the molecular mimicry and the escape from lysosomal degradation, which will be further explored. 2.2.1. Molecular mimicry The molecular mimicry theory is based on antigenic similarity between host tissue and infectious agents. Many bacterial pathogens mimic the function of host proteins to manipulate host physiology and cellular functions for their own benefit. In some cases, mimicry is achieved through virulence factors that are direct homologues of host proteins. In others, pathogens produced new effectors that, although having no obvious amino-acid sequence similarity to host factors, are revealed by structural studies to display mimicry at the molecular level [18]. The similarity between host and microbial antigens may be extremely harmful to host’s immune system. On the one hand, this mechanism may allow the organism to evade immune elimination and survive in the host. On the other hand, presence of common epitopes shared by microbial and host antigens may lead to the expansion of self-reacting lymphocytes and the induction of autoimmune phenomena, since the resultant antibodies act against both the foreign organism and the similar autoantigens [19, 20]. Molecular mimicry has been described as the main postulated mechanism by which infectious agents may break immunological tolerance and cause autoimmune disease [21]. Streptococcus pyogenes is one of the many pathogens that use molecular mimicry as a survival strategy. This important Gram-positive pathogen escapes the host immune system by expressing, namely, the group A carbohydrate N-acetyl glucosamine (GlcNAc), M protein and the cysteine protease streptococcal pyogenic exotoxin B (SPE B). GlcNAc and M protein have been shown to structurally mimic cardiac myosin, tropomyosin and vimentin [22-24], while anti-SPE B antibodies cross-react with host endothelial cells [25]. Yersinia spp also use this strategy, expressing virulence factors that target Rho GTPases in order to manipulate host cytoskeletal structure, and thereby evade the host’s immune system. Yersinia virulence factor YpkA, in addition to its host-like serine/threonine kinase activity, possesses additional host mimicry by harbouring key functions of the RhoGDI proteins, allowing host cytoskeleton disruption [26]. 2.2.2. Lysosomal degradation escape When a bacterium enters the host organism, macrophages, neutrophils and/or DCs, the so-called phagocytes, are attracted by a variety of substances generated in an immune response and move toward the infection site (chemotaxis) to engulf bacteria and other debris. Once internalized, the usual next step for a phagocytosed bacterium is to be channelled into the destructive environment of the lysosomes [27]. However, several strategies are used by bacteria to avoid lysosomal degradation, namely the inhibition of chemotaxis process, escape of phagocytosis process/elimination of phagocytes and prevention of phagosome-lysosome fusion. 2.2.2.1. Chemotaxis inhibition Some bacteria, such as Staphylococcus aureus, Bacillus anthracis, Streptococcus pyogenes and Clostridium perfringens can inhibit the chemotaxis process through the production of different toxins. These toxins prevent the phagocytes migration from the circulation to the site of infection and the consequent bacterial detection and phagocytosis. Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by several strains of Staphylococcus aureus and acts as a potent and specific inhibitor of neutrophils and monocyte chemotaxis CHIPS [28]. In Bacillus anthracis, this survival strategy is likely to be mediated by the adenylate cyclase activity of the component edema factor (EF) of anthrax toxin [29]. Even though EF is produced in an inactive form by the pathogen, its activation occurs upon contact with a heat-stable eukaryotic cell material. Once inside phagocytes, the adenylate cyclase activity of EF induces an increase of intracellular levels of cAMP, which in turn inhibits chemotaxis and improves bacterial growth during infection [30]. Streptococcus pyogenes and Clostridium perfringens produce, respectively, streptolysin and θ toxin that have been reported as repellents of neutrophils chemotaxis [31, 32]. 2.2.2.2. Phagocytosis escape/ Phagocyte elimination Important pathogens, such as Listeria monocytogenes, Shigella flexneri, Staphylococcus aureus and Pseudomonas aeruginosa employ different strategies to avoid lysosomal degradation that include escaping phagocytosis and/or release of toxins that are lethal to phagocytes. Listeria monocytogenes, for instance, expresses a pore-forming protein (listeriolysin O) that is able to create pores in the phagosomal membrane, which ultimately cause the disruption of the phagosomal membrane in a manner dependent on two different Listeria-encoded C-type phospholipases [33]. Once pores are formed, bacteria migrate from the phagosome into the cytosol where they are able to grow and divide. Once

1688

© FORMATEX 2013


inside the cytosol of infected cells, Shigella flexneri and Listeria monocytogenes coat themselves with a complex of proteins that actively induce actin polymerization/depolymerization. This actin polymerization allows bacteria to be pushed through the cell membranes into the neighbouring cells and therefore to avoid the host response mechanisms, such as complement and antibodies, which are only effective extracellularly [34]. Staphylococcus aureus can avoid destruction by neutrophils by releasing toxin leukocidin AB [35, 36]. This toxin is a member of the β-barrel pore-forming family and form octameric pores in the plasma membrane of the target cells [37], which result in osmotic imbalance and consequent cell death [35, 36]. Another example of pathogens that eliminate phagocytes as a strategy to escape the immune defense of the host is Pseudomonas aeruginosa. This pathogen produces toxins, such as phenazines [38] that induce an intermediate generation of reactive oxygen species (ROS) [39] and decrease of intracellular cAMP, followed by a rapid and overwhelming apoptosis of the neutrophil populations in vitro [38]. 2.2.2.3. Prevention of phagosome-lysosome fusion Another approach used by pathogens to avoid delivery to lysosomes after being phagocytosed is to prevent phagosome– lysosome fusion, a strategy used by pathogens such as Mycobacterium tuberculosis and Salmonella spp. This strategy not only ensures that bacteria can remain viable inside phagosomes, but also avoids the processing of bacterial antigens that would lead to activation of the immune system. Once inside phagosomes, pathogenic Mycobacteria can survive for extremely long times without being delivered to lysosomes. It seems that this pathogen recruits and retains the host protein TACO (tryptophan-aspartate containing coat protein) at the cytoplasmic face of the phagosome. This protein represents a component of the phagosome coat that is normally released prior to phagosome fusion with lysosomes. Active retention of TACO prevents the delivery to lysosomes, allowing Mycobacterium tuberculosis survival in phagosomes [40]. Several studies have shown that Brucella spp. also use the inhibition of phagosome-lysosome fusion as a mechanism of intracellular survival [41,42]. However, evidence suggests that this mechanism operates when invasion of the host cells is performed via lipid rafts [41]. Although it is still unclear, it has been shown that LPS O side chain of this pathogen is involved in the uptake of macrophages by targeting lipid rafts and results in non-fusion of phagosomes and lysosomes during the first hours after infection [42]. 2.2.3. Other strategies Host immune system usually recognizes antigens presented at bacteria cell wall. Some microorganisms release these antigens from their cell wall into the bloodstream, where will meet and eventually bind to correspondent antibodies, thus rendering that humoral immunity ineffective against those bacteria [43, 44]. Diverse strains of bacteria become encapsulated in order to evade the immune system, as their non-encapsulate form promotes complement activation as well as T cell commonly mediated immune response. It is possible to identify encapsulated bacterial strains that have been associated to clinically important infectious in humans, such as Neisseriae meningitidis, Penumococci and Haemophilus Influenzae. These bacteria can be responsible for meningitis, sepsis, being the latter two common causes of pneumonia and secundary bacterial lung infectious [2]. 2.3. Sphingolipids role in host-microbial pathogen interaction Sphingolipids have recently emerged as an important class of lipids involved in a variety of biological processes [45], including bacterial invasion of the host cells (e.g., Pseudomonas aeruginosa and Neisseriae gonorrhoeae), aiding on phagosomal maturation and promoting the fusion between phagosomes and lysosomes that result in bacterial killing (e.g., Listeria monocytogenes) [46]. Even though sphingolipids seem to be important players in controlling a number of bacterial infections, it has also been described that these lipids may indeed potentiate bacterial infections (e.g., Mycobacterium avium) [46]. This apparent contrasting function of sphingolipids is however dependent on the type of both the host cell and bacteria. Invasion of the host immune system cells, epithelial cells or fibroblasts by Pseudomonas aeruginosa and Neisseria gonorrhoeae is correlated with the activation and translocation into the plasma membrane of an important enzyme in sphingolipid metabolism, the acid sphingomyelinase (aSMase), which is responsible for the hydrolysis of sphingomyelin into ceramide [22, 47]. Ceramide, an important bioactive sphingolipid, is known to cause extensive alterations on membrane biophysical properties, including the formation of large ceramide-enriched domains, known as ceramide-platforms [47]. These domains seem to be important for the internalization of bacteria. The mechanism by which these lipid domains regulate bacteria internalization is yet to be resolved. Nevertheless, several lines of evidence show that blocking ceramide formation, either by genetic deficiency or pharmacological inhibition of aSMase, prevents bacterial internalization, death of the infected cells and controlled release of cytokines [46, 48, 49]. It has also been described that ceramide-platforms are involved in JNK-dependent apoptosis through the activation of NADPH oxidase and consequent derived ROS [46].

© FORMATEX 2013

1689


Listeria monocytogenes and Eschericia coli are two types of bacteria able to invade the host cell in a manner independent on the formation of ceramide-platforms [50]. However, activation of aSMase and formation of ceramide are required for phagosomal maturation in Listeria monocytogenes-infected cells. It has been shown that aSMase deficiency results in phagosomal maturation defects, perturbation of the lysosomal fusion process and decreased degradative capacity of the bacteria within the endo-phagosomal compartment. This compromises the effective killing of the bacteria and allows their escape into the cytosol [50-52]. One of the most striking features of the pathogenic Mycobacteria, such as Mycobacterium avium, is their ability to reside and replicate within macrophages, by preventing the phago-lysosomal fusion and inhibiting macrophagemediated killing mechanisms [46, 53]. These infected macrophages form large granulomas and multinucleated giant cells. The formation of such cells seems to be mediated by aSMase [50]. It was shown that aSMase genetic deficiency prevents the formation of multinucleated giant cells and conferred resistance to lethal infections promoted by these bacteria [50]. Other studies suggest that sphingolipids such as sphingomyelin, ceramide, sphingosine and sphingosine1-phosphate potentiate pathogenic mycobacteria (e.g., Mycobacterium avium and Mycobaterium tuberculosis) killing by stimulating actin nucleation and phagosomal maturation in infected macrophages [53].

3. DCs in innate and adaptive immunity 3.1. Subsets of DCs DCs represent a heterogeneous cell population derived from hematopoietic stem cells in the bone marrow [54] and are delivered to the most peripheral tissues through the blood. These APCs are classified according to their developmental origin, the cluster of differentiation expression, cytokine activators, surface antigens, functional capacity, anatomical location, and final outcome of immune response [55, 56]. Differentiation of DCs occurs by two specialized lineage models and it is divided into two major populations, referred to as myeloid DCs (mDCs) and plasmacytoid DC (pDC) [55-57](Figure 1). The mDCs are further subdivided in Langerhans cells (LCs) that are found in epidermis, oral, respiratory, and genital mucosa; interstitial, dermal or submucosal DCs (different names according to their anatomic location) [58, 59] and myeloid DCs. The plasmacytoid pathway, which generates only one major subset (the plasmacytoid DCs) was primarily found in blood and lymphoid organs [60]. Circulating pDCs are strong inducers of the innate immune response against viruses, secreting high amount of type I interferons (IFNs) [61, 62]. Despite the differences in subtypes, they all express high levels of MHC molecules [56].

Hematopoiet ic stem cells Myleoid precursor

Plasmacytoi d precursor

Monocyte

Langerhans DCs

Interstitial DCs

Myleoid DCs

Plasmocytoi d DCs

Fig. 1 Dendritic cells arise from myleoid and plasmacytoid pathways.

For all DC subtypes there are three stages of differentiation: DC precursors, immature DCs (iDCs), and mature DCs. Precursors and iDCs continuing arise in bone marrow and through the blood are delivered to peripheral tissue where they already have all the typical features of mature DCs [16]. The geographical localization of the DC subsets in secondary lymphoid tissues is distinct. Myeloid derived DC mainly migrate to or reside in the marginal zone (a primary entry point for blood-born antigens), whereas the lymphoid

1690

© FORMATEX 2013


DC mainly reside in the T-cell areas. It is now well appreciated that the DC subset, its maturation state and the microenvironment or type of pathogen that DC encounters in the periphery, determine the type of immune response that is induced, ranging from a Th1 or Th2 response to immune tolerance [63]. The follicular DCs are another type of DCs. They do not arise from hematopoietic origin but probably from mesenchymal and they have different functions from the APCs, because they do not express MHC II molecules. They are exclusively located in lymph follicles and express high levels of membrane receptors for antibody that interact with B cells (11). 3.2. DCs interaction with pathogenic and non-pathogenic bacteria In the absence of ongoing inflammatory response, iDCs constantly circulate through peripheral tissues, lymph and secondary lymphoid organs. Their function is to scan peripheral tissues where they recognize pathogen (bacteria, viruses, or danger toxins) invading the body and eliminate it by one of the endocytic mechanisms: receptor-mediated or receptor-independent endocytosis [56, 64-66]. The high ability of iDCs to capture antigen is due to their strategic localization and high endocytic capacity [56, 67]. On the other hand they have low T-cell activation capacity. In the absence of infection, DCs remain in the immature state [56, 68]. Once the pathogen enters the body through the skin or mucosal barrier, the first response of the host defence is an innate immune response. That is an universal host protection, that is mediated by epithelial cells, fibroblasts, endothelial cells, and the specialized migratory immune cells natural killer cells (NK cells), granulocytes, macrophages and DCs [56, 69]. Soon after infection, innate immune system informs the adaptive immune system about the pathogen invasion. This critical task is mainly fulfilled by DCs by recognition of elements exclusively expressed on bacteria, viruses, and fungi, generally known as PAMPs. These PAMPs are detected by pattern recognition receptors (PRRs) expressed on APCs and are involved in the initiation, promotion and execution of immune response. The main PRR families are Tolllike receptors (TLRs) [70-72], nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic-acid inducible gene (RIG)-like receptors (RLR) and C-type lectin receptor (CLR) [9, 73, 74]. PAMP-PRR interactions lead to activation of innate immunity [75]. The TLRs detect microbes on the cell surface and in endosomes, while the RLRs and the NLRs detect microbial components in the cytosol. TLR family is the largest and best studied PRRs and includes 10 (TLR 1-10) different receptors in humans and 12 (TLR 1-9 and 11-13) in mice, respectively [56, 76]. Classification of TLRs is based on the type of the structural component PAMPs that they recognize, such as lipids, proteins and nucleic acids. Lipids, like lipopolysaccharide (LPS) from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria and lipoarabinomannan from mycobacteria, are recognized by TLR1, 2, 4 and 6. TLR5 recognizes bacterial globular protein, flagellin [9, 77], while nucleic acids from viruses and bacteria are detected by TLR3, 7, 8 and 9. Mice TLR7 and human TLR8, have similar homology and both can detect single stranded RNA (ssRNA) and imidazoquinoline-like molecules. Moreover, mice TLR7 can also recognize small interfering RNA (siRNA). Additionally, TRL9 can detect non-methylated CpG DNA motifs from bacterial or viral genomes and also non-nucleic acids, like hemozoin from the malaria parasite [9, 74, 78]. Mammalian TLRs can also be divided in two subfamilies, according to their cellular localization. The first subfamily expresses the TLRs on the cell surface where they can directly interact with ligands. This group includes TLR1, 2, 4, 5, 6 and 10 in humans and 11 in mice. TLR3, 7, 8, and 9 represent the other subfamily of TLRs, located in the membrane of the endosome, where they can recognize viral or bacterial nucleic acids [79]. Stimulation of TLRs can enhance proliferation, activation of CD4+ and CD8+ and also B cells. The RLR family appears to be the key cellular sensor of RNA virus infection. Once RLR activation, it leads to antiviral host response, like induction of IFNs and expression of proinflammatory cytokines and chemokines. This family comprises three elements: retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), and they are all located in the cytoplasm. RIG-I detects RNA marked 5’ triphosphates (5’ppp) and containing viruses’ short double stranded RNA (dsRNA), and also detects positive and negative strand RNA viruses, influenza virus, Hepatitis C virus. MDA5 binds long dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). LGP2 ligand properties are less well defined, but they serve as either a positive or a negative regulator of RLR signaling [75]. The NLR family is located in the cytosol and it also regulates the innate immunity against microbial pathogens. In mammals, NLRs are divided in four subfamilies, based on different N-terminal effector domains. The most well-known members are NOD1 and NOD2. NOD2 is present only in monocytes, macrophages, DCs and intestinal Paneth cells, while NOD1 is omnipresent. Both induce NF-κB, but they recognize different structural motifs from peptidoglycans (PGN) of bacterial cell walls. NOD1 is activated by PGN γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) found in all Gram-negative bacteria and certain Gram-positive bacteria, while NOD2 is activated by muramyl dipeptide (MDP), PGN present in all Gram-positive and –negative bacteria [80-82]. Other PRRs commonly expressed by APCs are CLRs. CLR family is highly specific in recognizing and internalizing saccharides, like D-mannose, L-fructose, and N-acetylglucosamine of glycosylated self-antigens and pathogens. After antigen uptake and bounding on MHC I or II molecules, specific response can be induced, either by CD4+ or CD8+ Tcells [83, 84]. On the other hand, antigens that target mannose receptors are presented exclusively by the crosspresentation pathway. They target CD8+ T-cells through MHC I [9, 85].

© FORMATEX 2013

1691


3.3. Intracellular and intercellular signalling pathways in DCs function DCs have great capacity to orchestrate the host’s immune response to invading pathogens. iDCs digest the antigens into small fragments of peptides, present those on their surface and in this process become mature DCs. The fragments displayed on the surface membrane of the mature DCs are bound to the MHC class I or MHC class II, and the MHCantigen complex is presented to naïve T lymphocytes (CD8+ or CD4 + T cells) at lymphoid organs [64, 66, 71]. Through these maturation processes, DCs form a crucial link between innate and adaptive immunity and are essential for the development of antigen-specific immune responses. Comparing to iDCs, mature DCs show a decrease in their phagocytic capability, an augment in their efficacy to present processed antigens in the context of MHC molecules, and consequently an improved capability to activate T cells. Thus, mature DCs migrate from the sites of antigen capture to T-cell regions of draining lymph nodes, where they contact naïve or memory T cells and initiate a specific immune response [86-88]. During their migration, DCs express antigens that have been processed and present them on the surface by one of three pathways: through MHC class I, MHC class II or lipid antigen-presenting cluster of differentiation 1 CD1 molecules [6, 89]. The type of immune response is strongly dependent on the specific characteristics of pathogen, strength of signals, state of maturation of DCs, tissue and genetic factors. Usually, extracellular antigens that are taken up by DCs are fragmented by proteases in the DC endosomes, and antigen peptides are linked to the MHC class II and transported to the surface where they engage to activate CD4+ T helper cells. Alternatively, the exogenous antigens are also processed through a pathway called “cross-presentation” in which DCs, mostly mDCs, take up antigens and present them bound to MHC class I molecule. This mechanism activates CD8+ T cells in response to virus or tumour cells that do not infect APCs [90, 91]. Endogenously intracellular antigens are transported from the cytoplasm to the endoplasmic reticulum via the molecular complex transporter associated with antigen processing (TAP) and are then presented to the MHC class I molecule. As mentioned, this complex is recognized by CD8+ T cells [6, 90]. After a CD4+ T cell recognizes and binds to MHC class II molecule, the cell is activated and it starts to secret cytokines. They are important in activating B cells, CD8+ T cells and macrophages. CD8+ T cells, under the influence of CD4+ T cytokines, recognize an antigen-MHC I complex proliferate and differentiate into cytotoxic T lymphocytes (CTLs). Vital function of CTLs is monitoring the cells of the body and eliminating the one that express that specific antigen, like virus-infected or tumor cells [92]. For naïve T lymphocyte to become activated by DCs, different signals are required. First signal is the interaction between the T cell receptor (TCR) on the naïve T cell to the MHC molecule. Only those T lymphocytes that recognize the antigen presented by DCs become activated. The second signal is activated when interactions between costimulatory molecules, like CD80, CD86, and CD40 [92]. The third signal directly affects T cells, through cytokines and chemokines, like type 1 IFNs and IL-12, which are produced by DCs. These signals promote T cell migration, differentiation, survival and acquisition of effector functions [71, 93-95].

4. Nanoparticulate-based strategies to modulate immune response against microbial pathogens The mechanisms described in section 2 regarding microbial evasion and the host immune system complex mucosal network and their close cellular coordination have led to the development of several strategies focused on the induction of protective local immune responses through the administration of mucosal vaccines based on specific adjuvants or delivery systems [7, 12, 96]. The major advantage of those mucosal vaccines is their ability to simultaneously induce systemic and local protective immune responses, through the production of antigen-specific antibodies and the induction of cellular immune responses [97-99]. Since the introduction of the concept of vaccination with cowpox virus in 1796 by Edward Jener, vaccines have had a great impact on global health with the control of many infectious diseases and the eradication of smallpox [100, 101]. Despite the remarkable success of vaccination, the current approved vaccine modalities still find difficulties in inducing protection and constructing the right presentation of antigens for some infectious diseases, mainly caused by intracellular pathogens such as Mycobacterium tuberculosis and Salmonella enterica [102, 103]. For many years it was accepted that immune responses that must be elicited against extracellular and intracellular bacterial pathogens were devoted to the so-called Th2/Th1 paradigm, where Th1 cells protect the host from intracellular pathogens trough the induction of a cellular immune response and Th2 cells protect against extracellular pathogens through the stimulation of antibody production [104]. For the majority of the extracellular pathogens (Fig. 2A), protective immunity depends in fact on the presence of antibodies that are able to: (i) bind toxins and physically block their interaction with host cells; (ii) have a bactericidal effect mediated by the activation of the classical pathway of complement activation; or (iii) opsonise bacteria that are then ingested by macrophages [105]. In the case of intracellular bacterial pathogens (Fig. 2B), the generalizations are not so easy to apply. It is consensual that a cellular immune response, where CTL derived from CD8+ T cells play the most preeminent effector role, is essential for protection against intracellular bacterial pathogens [106]. Even though, it would be simplistic to consider that a cellular immune response should be the only concern of a vaccine against an intracellular bacterial pathogen since it seems that a synergy of different responses might be needed

1692

© FORMATEX 2013


to provide the adequate protection [107]. The role of antibodies in the protection against intracellular bacterial pathogens is not negletable at all. Antibodies have been shown to provide protection against intracellular bacterial pathogens by exerting a significant immunoregulatory effect on T-cell immunity and, in some cases, through the interaction with the pathogen before it enters target cells, during cell-to-cell spread of the pathogen or even inside the cell after membrane crossing [108, 109]. The great difficulty in developing such a vaccine is the identification of a single component (or a limited pool of components) that provides high levels of protection when used as an immunogen. Instead of a single gene product, such as a toxin with a major role in the pathogenesis, intracellular bacterial pathogens are often equipped with a battery of virulence factors with high complexity and limited visibility to the immune system, which are generally not good targets for antibody neutralization [110-112]. The selection of the best candidate antigens requires the application of distinct methods as, for example, the identification of antigens deeply involved in pathogenesis, bacterial survival or conserved sites of vulnerability between strains [113]. A multiplicity of strategies is currently under investigation (reverse vaccinology, codon deoptimization, immune refocusing, DNA shuffling, gene delivery by invasive bacteria, DNA plasmids, fusion proteins, cellular vaccines and innovative adjuvants, to name a few), which promise to contribute to the mitigation of some vaccine-missing bacterial infections and to the increase of efficacy of the currently approved vaccines [114-121].

Fig. 2 Desired immune responses against bacterial pathogens: a) once internalized by DCs, extracellular bacterial pathogens are digested and antigens are processed and presented through MHC class II complexes to CD4+ T cells. A Th2 profile is favoured, which results in the activation of B cells and secretion of antibodies against bacteria that can act through direct bacteria neutralization, complement activation and/or signalling bacteria to phagocytic cells through opsonization; b) intracellular bacterial pathogens seem to need a much more complex immune response with the involvement of cellular and humoral branches of the immune system. Bacterial antigens are presented through MHC class I to CD8+ T cells inducing the differentiation of cytotoxic T lymphocytes, which are the main effector cells in bacteria-infected cell lysis. CD8+ T cells can also develop a memory phenotype that will allow a prompt response in the case of a second infection. The presentation of bacterial antigens through MHC class II to CD4+ T cells is crucial for the enhancement of CTL functions through the auxiliary action of Th cells. Th1 cells enhance CTL functions through the secretion of cytokines (such as Il-2, Il.-12 and IFN-γ) and Th2 stimulate the secretion of antibodies by B cells. The recruitment of cells of the innate immune system, such as natural killer (NK) cells, granulocytes and macrophages, plays also an important role in the immune response against both extracellular and intracellular bacteria.

Vaccines currently under development aim to mimic the immune response stimulated by the natural infectious agent in order to induce a protective and specific immunity against the disease-causing pathogen. However, even that killed or live attenuated microorganisms and bacterial toxoids are highly effective in the induction of effector and memory immune responses, related safety issues have driven contemporary vaccine to be preferentially based on defined and purified recombinant subunit proteins [100]. It is well known that vaccines should comprise three critical elements: an antigen, an adjuvant, and a delivery system [122]. Antigens of different natures (proteins, peptides, lipids, etc) allow the development of a specific response by the adaptive branch of the immune system while adjuvants are able to activate the innate immune system. For many of bacterial pathogens it is necessary to use a mixture of subunits to elicit a protective response that is comparable to that elicited by a live attenuated vaccine [105]. Moreover, it is known that antigens and adjuvants must act concomitantly in order to induce a coordinated and effective immune response and the administration of an isolated antigen can induce tolerance to this specific entity [123]. Also, antigen immunogenicity and the profile of the elicited immune response can be manipulated by its combination with an adjuvant. Given that, a delivery system must be used to ensure a coordinated action of antigens and adjuvants in the activation of both innate and adaptive immune systems. As a result, the efficacy of a vaccine formulation can be considerably improved by the development of delivery systems able to protect and deliver antigens and adjuvants [124]. This section will focus in the use of polymeric NPs as vaccine delivery systems and their role in immunomodulation. Immunomodulation consists in the application of several strategies, such as the use of new materials, molecules and carriers to potentiate attenuate or simply modify immune responses to different pathologic and/or physiologic conditions [90]. Being the most professional APCs, DCs are preferential targets for immunomodulation since they

© FORMATEX 2013

1693


coordinate the downstream events of the immune response establishing a bridge between the innate and adaptive immune system [125]. As mentioned before, iDCs located in peripheral tissues, function as sentinel cells of the immune system by constitutively internalizing enormous quantities of solute upon detection of a maturation stimuli [87, 126]. Specific features of pathogens such as size, shape and surface molecule organization seem to be key factors for their recognition and internalization by DCs [127, 128]. Particulate vaccines, such as whole cell vaccines, virosomes, viruslike particles or antigens formulated in particulate adjuvants (liposomes, micro- and NPs), are able to provide an improved interaction with DCs due to their high surface area, which can provide electrostatic or receptor-interactions [129]. From all the classes of particulate vaccine delivery systems, NPs, especially those made of biodegradable and biocompatible polymers, have attracted special attention [127, 128]. In the 1970s, synthetic biodegradable polymers have started to be increasingly used for different biomedical applications in order to avoid the high costs, lack of reproducibility and questionable purity associated with natural polymers [130]. Synthetic polymers can now be easily produced with the uniformity required by regulatory entities for their application in final dosage forms, drug delivery systems (DDS) and in implantable devices [131]. Poly(amides), poly(aminoacids), poly(alkyl-α-cyanoacrylates), poly(esters), poly-(orthoesters), poly(urethanes), and poly(acrylamides) are some examples of synthetic polymers that have been used for biomedical applications [132]. Among them, the thermoplastic aliphatic polyesters are by far the most explored because of their excellent biocompatibility, biodegradability, and mechanical strength, which has resulted in a long history of their use in sutures, bone screws, tissue engineering scaffolds and DDS [133]. These polymers are degraded by a chemical hydrolysis reaction of ester bonds in their backbones resulting in the production of the original monomers, many of which are easily metabolized through the Krebs cycle and physiologically eliminated [134, 135]. Aliphatic polyesters are bulkeroding polymers, meaning that erosion happens throughout the polymer bulk and not exclusively from the surface inwards [136]. The reaction can be catalyzed by acids or bases and the source of the catalyst can be external (nonautocatalytic reaction) or internal (autocatalytic reaction) when it happens by the carboxylic acid end groups of the polymer chain [137]. The degradation rate is related to the monomer constitution of the polymer. In the case of PLGA, higher content of glycolic acid, which is slightly more hydrophilic than lactic acid, leads to increased hydrolysis rates [138]. Simple formulation methods can be used to modify aliphatic polyesters in order to design carrier systems able to deliver a variety of drug classes such as peptides, proteins, and micromolecules. Most importantly, the Food and Drug Administration (FDA) and European Medicine Agency (EMA) approve them for drug delivery in humans [9, 139, 140]. Aliphatic polyesters can be synthesized by polycondensation of a hydroxy acid or a diol and a diacid [141], ring opening polymerization (ROP) of lactones and other cyclic diesters such as lactide and glycolide [142, 143] or by enzymatic polymerization under mild conditions avoiding the use of toxic reagents and with the possibility to recycle the catalyst [144, 145]. The aliphatic polyesters mainly used for biomedical applications are those derived from the monomers glycolide/glycolic acid (GA), lactide/lactid acid (LA), β-butyrolactone (β-BL), ε-caprolactone (ε-CL), 1,5dioxepan-2-one (DXO) and trimethylene carbonate (TMC). They can be classified as homopolymers or copolymers depending on their homogenous or heterogenous composition on repeating units, respectively [142]. The homopolymers polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL) and the copolymer poly(lactic-co-glicolic acid) (PLGA) are by far the most explored aliphatic polyesters for DDS formulation [146-149]. PLGA, one the most successfully developed biodegradable polymers for biomedical applications, has been widely used to formulate different biodegradable devices, such as microparticles (MPs) and NPs, implants and miscellaneous devices [150]. Biodegradable PLGA particles have been investigated for sustained and targeted/localized delivery of various agents, including hydrophilic and hydrophobic drugs, proteins and peptides, plasmid DNA and siRNA, because of their ability to protect these agents from the aggressive in vivo environment [151, 152]. It has also been extensively tested as vaccine particulate delivery systems to encapsulate a variety of antigens such as proteins, peptides, lipopeptides, cell lysates, viruses or plasmid DNA have been successfully formulated in PLGA NPs [153-158]. Several advantages have been recognised on the application of PLGA NPs as vaccine delivery systems/immunomodulatory tools (Table 1), some of them are transversal to NPs of other nature. Several studies have demonstrated that NPs could increase the internalization of antigens and adjuvants by DCs providing better immune responses than those obtained with the soluble counterparts [159]. Apart from the higher uptake by DCs, NPs also provide protection to antigens and adjuvants, including proteins, peptides, and nucleic acids, from degradation in biological fluids [160]. Some molecules used as vaccine adjuvants, such as TLR ligands, can cause a life-threatening septic shock-like state and lead to organ dysfunction [159]. Here, NPs can also have a roll in increasing vaccine safety since lower doses of adjuvants are needed and their systemic distribution is more restricted [161]. NPs composed by aliphatic polyesters are able to confer a controlled release profile to the encapsulated agents with a tunable rate of release through the manipulation of physicochemical properties of the polymer, such as the molecular weight, or by copolymerization with other polymers [159]. Cargo release from aliphatic polyester particulate delivery system is often described to happen in three phases: an initial phase with high rate of release (burst release phase) due to the release of adsorbed molecules to the particle surface; an intermediated slow phase of release of the molecules entrapped in the polymeric matrix (lag phase), which is closely related to the bulk degradation of the polymer; and a second phase with increased release rate (final release phase) [162, 163]. This release profile can be benefic for vaccine formulation since

1694

© FORMATEX 2013


the dose of antigen necessary to stimulate the secondary response is much lower than that required for evoking a primary response [164]. As so, aliphatic polyester-based particulate vaccine delivery systems can contribute to the development of single-injection vaccines where the burst effect can mimic the priming dose and the sustained release can simulate the effect of booster doses required for current vaccination programs [165]. Another strategy is to conjugate particles of different sizes or polymers with different degradation rates (for instance, PLGA with different lactide:glycolide ratios), to provide different release rates of antigens and adjuvants [166]. It has also been verified that particulate formulation of different sizes can deliver antigens to separate subsets of DCs, inducing Th1 and/or Th2 responses [167]. A sustained release of adjuvants, such as TLR ligands, is essential to properly stimulate DCs for longlasting proinflammatory cytokine production and to reverse the immunosuppressive action of regulatory T cells (Treg) [168, 169]. Additionally, some particulate systems, particularly those constituted by PLGA, have been shown to escape from early endosomes to the cytoplasm potentiating the proteasome-dependent processing of encapsulated antigens and cross-presentation through MHC class I [170, 171]. Table 1 Main advantages and disadvantages of PLGA as a particulate vaccine delivery system component. Advantages • Biodegradability and biocompatibility; • Approval for parenteral use by regulatory authorities around the world; • Commercially available with different physicochemical properties (several molecular weights and lactide:glycolide ratios); • Cargo release profile can be tailored by selecting PLGA polymers with the appropriate properties; • Protection of encapsulated biomolecules from in vivo degradation in the blood stream, gastro-intestinal tract and nasal mucosa; • Delivery of antigens to be presented by both MHC class I and MHC class II pathways; • Deliver more than one agent concomitantly in the same particulate delivery system (eg. several antigens or co-encapsulation of antigen and adjuvant) increasing the probability of coordinate and synergistic action; • Potential for vaccine delivery through alternative routes od adiminstration (topical, pulmonary, ocular or even oral); • PLGA particles are able to induce strong T cell responses containing very low doses of antigens and adjuvants, allowing the use of lower doses of these molecules minimizing the potential side effects often associated with the use of adjuvants; • Blending or co-polymerizing with other materials; • Can be stably stored and easily scaled up for pharmaceutical manufacture; Disadvantages • Environment acidification upon degradation compromising cargo-stability; • Risk of denaturation of the incorporated biomolecules by the organic solvents and/or shear stress used during particle formulation; • Low encapsulation efficiencies of some macromolecules; • Lack of suitable functional groups for efficient covalent bioconjugation of bioactive ligands or the adsorption of negatively charged therapeutics such as nucleic acids; • Required sterilization for clinical use; • Potential expensive DDS. From [6] Silva JM. et al. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release 2013 Mar 21;168(2):179-199. Reprinted with permission of Elsevier”

The fact that polymeric NPs, additionally to their vaccine delivery function, act as vaccine adjuvants by themselves (i.e., without the addition of an immunomodulator molecule) is not consensual [172]. PLGA NPs have been successfully tested in adjuvant-free vaccine for anthrax [165]. It seems that polymer/surfactant and particulate systems can induce complement activation in a complex process dependent on inter-related physicochemical factors such as hydrodynamic size, morphology, composition and surface properties [173-175]. A study on PLGA micro- and NPs demonstrated potent inflammatory stimulus after the uptake process with NF-κB translocation to cell nucleus and further pro-inflammatory cytokines production [176]. However, empty polymeric NPs are normally referred to have only a limited capacity to elicit antibody secretion or to induce a cellular immune response against them. It has also been demonstrated that empty PLGA MPs with or without polycationic coatings did not induced significant maturation neither affected the capacity of DCs to respond to a maturation-inducing cocktail afterwards [177]. Even that the negligible adjuvant properties are pointed out as one of the disadvantages of particulate aliphatic polyester for vaccine delivery systems, intrinsic adjuvant effects can be considered in a certain way, and some are mentioned on Box 1. These intrinsic adjuvanticity of NPs is deeply connected to their repetitive antigen display, their pathogen-like size, the depoteffect and their capacity to target the immune system, not only at the tissue and cellular levels but also at the subcellular level [124, 178]. NPs can provide direct intracellular access, facilitating engagement of the intracellular receptors such as the TLR3, 7, 8 and 9, improving the efficacy of vaccine adjuvants [179]. Also, NPs can direct antigens to different routes of presentation (MHC I or MHC II) [180]. The internalisation mechanisms and intracellular trafficking pathways followed by NPs can influence the immunomodulatory effects and different profiles of immune response can be obtained [90, 181]. The decision of which endocytic mechanism(s) and intracellular pathway(s) will be followed by NPs is complexly influenced by many

© FORMATEX 2013

1695


physico-chemical factors, such as size, charge, geometry, chemical constitution, adhesion energy, surface tension of membrane and bending rigidity ratio between nanocarrier and membranes [127, 128]. Understanding the interdependence of these factors on the cellular internalisation and intracellular trafficking and fate is extremely important. Some considerations about these factors are presented on Table 2. These physicho-chemical factors are also the basis of passive targeting of NPs to preferential tissues and cell types [129]. Box 1 Inherent adjuvanticity of aliphatic polyester particulate delivery systems for vaccines. • • • •

Ability to protect immunogens from rapid degradation and clearance; Efficient internalization by APCs; Extracellular reservoirs of immunogens due to sustained release occurring for lengthy periods (depot effect); Intracellular reservoirs of immunogens in APCs after particle uptake so that intracellularly released antigens can be presented by the APCs to specific T cells over extended periods of time; • Capacity to directly migrate to lymph nodes after peripheral administration (for particles < 200 nm); • Codelivery of several immunogens, being either several antigens or antigens and adjuvant molecules; • Capacity to induce presentation of antigens through both MHC class I and MHC class II complexes. From [6] Silva JM. et al. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release 2013 Mar 21;168(2):179-199. Reprinted with permission of Elsevier” Table 2 Considerations about the influence of physicochemical factors of NPs as vaccine delivery systems. Size • No evidence of one optimal size for internalization – DCs can process any antigen within pathogens’ dimensions, from viruses (20-100 nm) to bacteria and even cells (in the micrometer range) [129]; • Influence on endocytic mechanism: • virus-sized particles (20–200 nm) are usually taken up via classic receptor-mediated endocytosis (clathrin-dependent) (particles < 200 nm) [182] or endocytosis through caveolae (particles within 50–100 nm) [183] and tend to generate a virus-like immune response with activation of CTL and Th 1; • larger sized particles (> 0.5 µm) are internalized mainly via macropinocytosis or phagocytosis and generates preferentially a bacteria-like immune response with activation of Th2 and secretion ofantibodies [184, 185]; • Influence on membrane wrapping time and diffusion rate: smaller NPs have higher surface curvature that restricts the binding of NPs to cells but the wrapping time of membrane over NPs and the receptor diffusion rate are higher [186, 187]; • Influence on the route of administration of the NP vaccine: • subcutaneous (s.c.) and intramuscular (i.m.) injections are the most traditional routes for immunization which are suitable for almost any type and size of particles [188]; • for mucosal administration, since the mucus consists in a physically crosslinked, viscoelastic hydrogel with mesh sizes of ~100 nm, NPs > 100 nm will have more difficulties in cross the mucus and reach the MALT [189, 190]. • for the targeting of lymph nodes trough dermal administration, only NPs < 200 nm can efficiently enter the lymphatic system and reach the nodes directly within hours after injection,. NP > 200 nm do not efficiently enter the lymphatic system unless they are associated with APCs and will take approximately 24 hours to arrive in the lymph nodes [191, 192]. Charge • Positively charged NPs: higher extent of internalization, apparently as a result of the ionic interactions with the negatively charged cell membranes by non-specific adsorptive endocytosis, which is also the reason for higher toxicity of positively charged NPs [193-195]; • Some of positively charged NPs could escape from lysosomes after being internalized and exhibit perinuclear localization; • Negatively charged NPs and neutral NPs prefer to colocalize with lysosomes [196, 197]. • Charge is also important for formulation stability (neutral NPs tend to aggregate). Chemical constitution/hydro- and lipophilicity/surface chemistry • Surface chemistry is an indispensable manipulation for purposes like functionalization, reducing surface reactivity, reducing toxicity, or enhancing stability [198]. • Surface chemistry influence the nature and constitution of the “protein corona” that is associated with NPs in biological fluids: • Surface PEGylation minimize protein adsorption and consequently reduce recognition of NPs by the mononuclear phagocytic system [199]; • more hydrophobic surfaces allow higher protein adsorption to NP surface [200]; • hydrophilicity/hydrophobicity can have an impact on the extent of cellular uptake and intracellular trafficking and fate [201]. Shape • NP shape can influence the accumulation and concentration rates of the NPs in a cell: spherical-shaped NPs have higher cellular uptake comparing to sheet-shaped or nanorod-shaped counterparts [202-204].

NPs can also be actively targeted to specific cells and tissues by surface functionalization with ligands directed against cell surface receptors that function as a gateway to the immune system [205]. Different classes of ligands such as peptides, antibodies, proteins, polysaccharides, glycolipids, glycoproteins, and lectins can be used to functionalize

1696

© FORMATEX 2013


NPs [206]. These ligands can be specific for cell surface receptors of defined cell types of the immune system and enhance cellular specific internalization or even contribute to the enhancement of the immunogenicity of the particulate vaccine, by providing an intrinsic “danger signal” that induce the activation of innate and adaptive immune mechanisms [207-209]. An important issue for vaccine design is the consideration of the ligands carried by a number of pathogens that can interact with receptors on cells of the immune system. As mentioned before, APCs possess a broad spectrum of receptors (the PRRs) that recognize conservative elements exclusively expressed on pathogens (the PAMPs) and are involved in the initiation, promotion and execution of immune responses [73, 74, 210]. Recognition of PAMPs by PRRs induces activation of APCs by triggering intracellular signalling pathways that lead to the induction of inflammatory cytokines, chemokines, interferons and upregulation of co-stimulatory molecules [211]. Aliphatic polyesters-based particulate delivery systems have been tested as carriers for antigens and adjuvants for prophylactic vaccination strategies (Table 3). In recent studies, our group demonstrated that polymeric micro- and NPs were able to influence Streptococcus equi enzymatic extract presentation by the immune system, inducing a mucosal and balanced Th1/Th2 immune response crucial for protection against S. equi infection [212]. Those studies focused mainly on mucosal immunisation, which is thought to be more appropriate for a respiratory infection such as equine strangles, while emphasizing the mucosal adjuvant properties of PCL nanospheres containing S. equi protein extract. Although some state that aliphatic polyesters homopolymers are quite limited in terms of conjugation possibilities, the use of these polymers in the formulation of particulate delivery systems broaden the possibilities of conjugation strategies of different classes of molecules. For instance, Florindo et al. (2009) tested PLA nanospheres as vaccine delivery systems for the delivery of the S. equi M-like protein (SeM) concomitantly with other molecules with adjuvant properties, such as spermine, oleic acid, alginate and glycol-chitosan [7]. Guo and Gemeinhart (2008) also demonstrated that chitosan can easily be adsorbed to PLGA NPs, yielding NPs with highly positive zeta potential, which are useful to increase the loading of negatively charged biomolecules, such as proteins, peptides and nucleic acids [213]. RothWalter et al. (2005) functionalized PLGA micropheres with Aleuria aurantia lectin taking advantage of the available surface carboxylic groups of the polymer for covalent coupling with the protein [214]. Table 3 Examples of aliphatic polyester particulate delivery systems applied to prophylactic vaccination against bacteria. Carrier PLGA NPs

Aim Application: single dose prophylactic vaccine against anthrax. Aim: improve currently approved anthrax vaccine in terms of boost requirements, adjuvant-associated side effects and stability by using PLGA NPs associated with the Protective Antigen Domain 4 (PAD4) from Bacillus anthracis.

PLA NS

Study design Design: Preclinical (mice), immune response evaluation, after i.p. immunizations. Median survival after challenge with anthrax spores. Groups: 1. PAD4-NP 2. PAD4 sol. 3. Blank-NPs

Application: horse protection from the infectious disease strangles.

Design: Preclinical (mice), immune response evaluation after i.m. immunizations.

Aim: evaluate the humoral and cellular immune responses induced by i.m. vaccination with Streptococcus equi enzymatic extract or purified recombinant S. equi M-like protein (SeM) entrapped in PLA nanospheres and the effect of including molecules with adjuvant properties such as spermine (SP), oleic acid (OA), alginate (ALG) and glycolchitosan (GCS)

Groups: 1. PLA-PVASeM 2. PLA-GCSSeM 3. PLA-ALGSeM 4. PLA-SPSeM 5. PLA-OASeM 6. PLA-PVASeM+CpG 7. SeM+CpG sol. 8. SeM sol.

© FORMATEX 2013

Outcome • The antibody titer induced by PAD4-NP was markedly higher than that elicited by the soluble antigen; • IgG titer increased 80 fold from day 14 to day 28 while with PAD4-NP while it was kept constant for PAD4 sol.; • PAD4-NP elicited a mixed IgG1 and IgG2a response, whereas PAD4 induced predominantly IgG1 immune response. • PAD4-NP induced higher and balanced IFN-γ and IL-4 levels while PAD4 sol. Induced very low levels of IFN-γ and medium levels of IL-4; • PAD4-NP immunized mice showed a median survival of 6 days with 11% survival, whereas PAD4 sol., Blank-NP and PBS immunized mice had a median survival of 1 day. • The humoral immune response induced by NS was markedly higher than that elicited by soluble antigens, isolated or co-admixed with CpG; • The IgG and IgG subtypes and cytokine titres indicated that nanospheres with GCS developed a more balanced Th1/Th2 response for both purified SeM and S. equi enzymatic extract proteins, although those induced by the pure antigen-entrapped particles were higher than the S. equi tested vaccines composed by total antigens entrapped in polymeric NS.

Ref. [165]

[215]

1697


PLA MPs and NPs

PCL MPs

PLGA NPs

Application: prophylactic vaccine for Salmonella typhi infections.

Design: Preclinical (mice), immune response evaluation, after i.m. immunizations.

Aim: improve immunogenicity of Vi polysaccharide antigen from Salmonella typhi by entrapping and delivering it using biodegradable polymer particles. Coentrapment of a carrier protein, Tetanus toxoid (TT), and Vi polysaccharide antigen in the same PLA particle was also tested. Higher surface density of Vi polysaccharide in NPs and MPs was also evaluated.

Groups: 1. MP(Vi) 2. NP(Vi) 3. MP(Vi+TT) 4. NP(Vi+TT) 5. MP(Vi)-higher density 6. NP(Yi)-higher density

Application: prophylactic vaccine for Brucella ovis.

Design: Preclinical (rams), immune response evaluation, after s.c.. immunizations. Pathological studies after challenge.

Aim: determine the efficacy of the previously studied PCL MPs loaded with higher doses of hot saline antigenic complex (HS); determine the protective efficacy of the same antigen complex incorporated in NPs methylvinylether and maleic anhydride (Gantrez®AN, ISP Corp.). Application: prophylactic vaccine for Chlamydia trachomatis.

Groups: 1. HS-PCL MPs 2. HS-NPs 3. Man-HS-NPs 4. Rev 1* 5. unvaccinated Design: In vitro (mouse J774 macrophages

Groups: 1. MOMP-NPs Aim: encapsuylate the major 2. MOMP sol. outer membrane protein (MOMP) of C. trachomatis and evaluate its capacity to induce Th1 cytokines and nitric oxide production in vitro. *Reference vaccine (Live attenuated strain B. melitensis)

• Immunization with particles entrapping Vi antigen elicited significantly higher anti-Vi IgM response than that observed from immunization with soluble Vi antigen; • Vi-entrapped NPs elicited a stronger IgG response and significantly higher memory antibody responses than Vi MPs; • Coentrapment of Vi-polysaccharide antigen and TT in both NPs and MOs resulted in lower anti-Vi and anti-TT antibody titers; • Upon challenge with S. typhi, groups primed with particulate formulations elicited stronger anti-Vi recall responses; • Surface density of Vi antigen on polymer particle surface was one a major determinant in eliciting higher anti-Vi polysaccharide antibody response; • The results indicate that polymer particle based vaccine delivery system improves the immunogenicity of T-independent antigens considerably from a single immunization dose. • All immunizations induced high levels of IgG but HS-NP allowed high levels for longer period; • All immunizations induced high levels of IFN-γ secretion that were maintain at least after 6 months after the immunization; • Imunized animals with NPs and MPs had in general less affected less lesions from infection.

• MOMP-NPs induced higher levels of IL6, IL-12p40 and NO then MOMP sol.; • manner; • MOMP-NP induction of IL-6 and NO was dose-dependent but not for IL12p40.

[216]

[217]

[218]

Polymeric NPs have demonstrated promising results as particulate vaccine delivery systems and several advantages over the currently available strategies. The field of particulate vaccine delivery systems is rapidly evolving and those based on aliphatic polyester polymers are closer to make a major contribution to improved vaccinology in the 21st century. The expectation is that the particulate vaccine delivery systems along with the recent developed techniques in detecting the most appropriate bacterial targets will be better at providing potent antigen-specific humoral and cellular immune responses and will allow next generation vaccines to be devised against a range of infectious diseases. With the development of many new vaccines, children immunization programs have to deliver a large number of inoculations, a burden for them and for the mothers [219]. Combining antigens is one of the solutions despite their technical, clinical, and regulatory difficulties. Nanoparticulate delivery systems can have an important role in combining several antigens in one vaccine. An aging population requires preventions such as influenza, pneumococcal, shingles, and regular booster. Polymeric NPs with a prolonged antigen and adjuvant release might also solve the problem of the need of regular booster for elderly. Nevertheless, several difficulties still need to be overcome. These difficulties are both related to the vaccine development process - need for validated methods to clinically evaluate new vaccine strategies; selection of most relevant antigens and epitopes; determination of the optimal target population, doses and schedules as well as with the particulate delivery systems – determination of whole body and cellular pharmacokinetics; stability; drug release rates and translation to clinic.

1698

© FORMATEX 2013


4.1. Translational aspects and regulatory requirements Current translational requirements for nanoparticulate-based systems in immunomodulation against microbial pathogens have to deal with aspects related to: (i) conventional approaches for translational research of antimicrobial medicinal products [220-222]; (ii) aspects related to immunomodulation looking at risk-benefit specific assessment and the toxicological relevance of activating a number of immunomodulatory pathways [223]; (iii) the importance of immunomodulatory effects of a number of particulate systems, either activating specific cascades [224], or potential deleterious effects through non-intended accumulation of synthetic polymers [225] or potential for generating a stronger immune response with non-intended systemic impact [226].

5. Concluding remarks and future prospects. CMIS constitutes a complex network responsible for an ultimate induction of a disseminated immune response when in contact with microbial pathogens that uses mucosal surfaces as their main portal of entrance and possess smart strategies to overcome host immune responses. Among the variety of cells involved in this coordinated phenomena, DCs represents the single most central player in all immune responses, both innate and adaptive. The recognition of microbial patterns can result in DC maturation, which in turn directly affects DC functions: immature DC efficiently take up antigen but are poor at activating T cells, whereas mature DC shut down antigen acquisition but up-regulate antigen presentation and the co-stimulatory machinery required for efficiently priming T cells. As so, innovative and effective strategies have risen in order to use advanced delivery systems to potentiate antigen recognition, capture and further presentation to T and B cells. The administration of those particulate systems through a mucosal surface constitutes a very efficient approach, as it will be able to induce not only a systemic, but also cellular and local protective immune responses. Acknowledgements The support by Fundação para a Ciência e Tecnologia, Ministério da Ciência e Tecnologia, Portugal (SFRH/BD/64295/2009, SFRH/BD/78480/2011, SFRH/BD/87591/2012, compromisso para a Ciência 2008 and PTDC/SAUFAR/119389/2010) and Fonds de la Recherche Scientifique Médicale (Belgium) is gratefully acknowledged.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

Brodsky IE, Medzhitov R. Targeting of immune signalling networks by bacterial pathogens. Nature cell biol. 2009;11:521-526. Baxt La, Garza-Mayers AC, Goldberg MB. Bacterial subversion of host innate immune pathways. Science. 2013;340:697-701. Bueno SM, Riquelme S, Riedel Ca, Kalergis AM. Mechanisms used by virulent Salmonella to impair dendritic cell function and evade adaptive immunity. Immunology. 2012;137:28-36. Tacken PJ, Figdor CG. Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines. Sem immunol. 2011;23:12-20. Ueno H, Klechevsky E, Schmitt N, Ni L, Flamar A-L, Zurawski S, et al. Targeting human dendritic cell subsets for improved vaccines. Sem immunol. 2011;23:21-27. Silva JM, Videira M, Gaspar R, Préat V, Florindo HF. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Control Release. 2013;168:179-199. Florindo HF, Pandit S, Lacerda L, Gonçalves LMD, Alpar HO, Almeida aJ. The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-epsilon-caprolactone-based nanoparticles. Biomaterials. 2009;30:879-891. Garg S, Sharma M, Ung C, Tuli A, Barral DC, Hava DL, et al. Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b. Immunity. 2011;35:182-193. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release. 2012;161:505-522. McGhee JR, Fujihashi K. Inside the mucosal immune system. PLoS biology. 2012;10:e1001397-e1001397. Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, Tucková L, Cukrowska B, Lodinová-Zádníková R, et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Let 2004;93:97-108. Garinot M, Fiévez V, Pourcelle V, Stoffelbach F, des Rieux A, Plapied L, et al. PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination. J Control Release. 2007;120:195-204. Andrews GP, Laverty TP, Jones DS. Mucoadhesive polymeric platforms for controlled drug delivery. Eur J Pharm Biopharm. 2009;71:505-518. Ugwoke MI, Agu RU, Verbeke N, Kinget R. Nasal mucoadhesive drug delivery: Background, applications, trends and future perspectives. Adv Drug Deliv Rev. 2005;57:1640-1665. Janeway CA, Medzhitov R. Innate immune recognition. Annual Review of Immunology. 2002;20:197-216. Foster N, Hirst BH. Exploiting receptor biology for oral vaccination with biodegradable particulates. Adv Drug Deliv Rev. 2005;57:431-450. Hornef MW, Wick MJ, Rhen M, Normark S. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunology. 2002;3:1033-1041.

© FORMATEX 2013

1699


[18] Stebbins Ce Fau - Galan JE, Galan JE. Structural mimicry in bacterial virulence. Nature.412:5. [19] Barnaba V, Sinigaglia F. Molecular mimicry and T cell–mediated autoimmune disease. The Journal of experimental medicine. 1997;185:1529-1534. [20] Hemmer B, Fleckenstein BT, Vergelli M, Jung G, McFarland H, Martin R, et al. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone. J Exp Med. 1997;185:1651-1670. [21] JK O, Croxford L, Miller S. Innate and adaptive immune requirements for induction of autoimmune demyelinating disease by molecular mimicry. Mol Immunol.40:1103-1109. [22] Fenderson PG, Fischetti VA, Cunningham MW. Tropomyosin shares immunologic epitopes with group A streptococcal M proteins. J Immunol. 1989;142:2475-2481. [23] Cunningham M, Antone S, Smart M, Liu R, Kosanke S. Molecular analysis of human cardiac myosin-cross-reactive B- and Tcell epitopes of the group A streptococcal M5 protein. Infect Immun. 1997;65:3913-3924. [24] Kraus W, Seyer J, Beachey E. Vimentin-cross-reactive epitope of type 12 streptococcal M protein. Infection and Immunology. 1989;57:5. [25] Luo Y, Chuang W, Wu J, Lin M, Liu C, Lin P, et al. Molecular mimicry between streptococcal pyrogenic exotoxin B and endothelial cells. Lab Invest 2010;90:15. [26] Prehna G, Ivanov M, Bliska J, Stebbins C. Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors. Cell. 2006;126:12. [27] Goldsby RA KT, Osborne BA, Kuby J. Immunology. San Francisco2002. [28] de Haas CJC, Veldkamp KE, Peschel A, Weerkamp F, Van Wamel WJB, Heezius ECJM, et al. Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent. J Exp Med. 2004;199:687-695. [29] Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci. 1982;79:3162-3166. [30] O'Brien J FA, Dreier T, Ezzell, Leppla S. Effects of anthrax toxin components on human neutrophils. Infect Immun. 1985;47:5. [31] Petit L, Gibert M, Popoff MR. Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 1999;7:104-110. [32] Lin A, Loughman J, Zinselmeyer B, Miller M, Caparon M. Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection. Infect Immun. 2009;77:11. [33] Goebel W, Kuhn M. Bacterial replication in the host cell cytosol. Curr Opin Microbiol. 2000 2009;3:49-53. [34] Cameron LA, Footer MJ, van Oudenaarden A, Theriot JA. Motility of ActA protein-coated microspheres driven by actin polymerization. Proc Natl Acad Sci. 1999;96:4908-4913. [35] Alonzo F, Torres VJ. Bacterial survival amidst an immune onslaught: the contribution of the Staphylococcus aureus leukotoxins. PLoS Pathog. 2013;9:4. [36] Vandenesch F, Lina G, Henry T. Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: a redundant arsenal of membrane-damaging virulence factors? Front Cellular Infect Microbiol. 2012 20120824;2:15. [37] Yamashita K, Kawai Y, Tanaka Y, Hirano N, Kaneko J, Tomita N, et al. Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components. Proc Natl Acad Sci. 2011;108:17314-17319. [38] Usher LR, Lawson RA, Geary I, Taylor CJ, Bingle CD, Taylor GW, et al. Induction of Neutrophil Apoptosis by the Pseudomonas aeruginosa Exotoxin Pyocyanin: A Potential Mechanism of Persistent Infection. J Immunol. 2002;168:18611868. [39] Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukoc Biol. 1993;54:283-288. [40] Ferrari G, Langen H, Naito M, Pieters J. A Coat Protein on Phagosomes Involved in the Intracellular Survival of Mycobacteria. Cell. 1999;97:435-447. [41] Watarai M, Makino S-i, Fujii Y, Okamoto K, Shirahata T. Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell Microbiol. 2002;4:341-355. [42] Porte F, Naroeni A, Ouahrani-Bettache S, Liautard J-P. Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages. Infect Immun. 2003;71:1481-1490. [43] Sanderson-Smith M, De Oliveira DMP, Ranson M, McArthur JD. Bacterial plasminogen receptors: mediators of a multifaceted relationship. J Biomed Biotechnol. 2012;2012;1-14. [44] Van Amersfoort ES, Van Berkel TJC, Kuiper J. Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock. Clin Microbiol Rev. 2003;16:35. [45] Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol. 2008;9:139-150. [46] Grassmé H, Becker K. Bacterial Infections and Ceramide. In: Gulbins E, Petrache I, editors. Sphingolipids in Disease. Handbook of Experimental Pharmacology. 216: Springer Vienna; 2013. p. 305-320. [47] Grassmé H, Riethmüller J, Gulbins E. Biological aspects of ceramide-enriched membrane domains. Progr Lipid Res. 2007;46:161-170. [48] Hauck CR, Grassmé H, Bock J, Jendrossek V, Ferlinz K, Meyer TF, et al. Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae. FEBS Lett. 2000;478:260-266. [49] Zhang Y, Li X, Carpinteiro A, Gulbins E. Acid Sphingomyelinase Amplifies Redox Signaling in Pseudomonas aeruginosaInduced Macrophage Apoptosis. J Immunol. 2008;181:4247-4254. [50] Utermöhlen O, Herz J, Schramm M, Krönke M. Fusogenicity of membranes: The impact of acid sphingomyelinase on innate immune responses. Immunobiol. 2008;213:307-314. [51] Utermöhlen O, Karow U, Löhler J, Krönke M. Severe Impairment in Early Host Defense Against Listeria monocytogenes in Mice Deficient in Acid Sphingomyelinase. J Immunol. 2003;170:2621-2628. [52] Schramm M, Herz J, Haas A, Krönke M, Utermöhlen O. Acid sphingomyelinase is required for efficient phago-lysosomal fusion. Cell Microbiol. 2008;10:1839-1853.

1700

© FORMATEX 2013


[53] Anes E, Kuhnel M, Bos E, Moniz-Pereira J, Habermann A, Griffiths G. Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria. Nat Cell Biol. 2003;5:793-802. [54] Diebold J. [Mononuclear phagocyte system. Morphology and function of the principal constituting cells]. Ann Pathol. 1986;6:3-12. [55] Kah-Wai L, Jacek R, Jacek T. Dendritic cells heterogeneity and its role in cancer immunity. J Cancer Res Ther. 2006;2:35-40. [56] Fajardo-Moser M, Berzel S, Moll H. Mechanisms of dendritic cell-based vaccination against infection. Int J Med Microbiol. 2008;298:11-20. [57] Robinson SP, Patterson S, English N, Davies D, Knight SC, Reid CDL. Human peripheral blood contains two distinct lineages of dendritic cells. Eur J Immunol. 1999;29:2769-2778. [58] Faith A, Hawrylowicz CM. Targeting the dendritic cell: the key to immunotherapy in cancer? Clin Exp Immunol. 2005;139:395-397. [59] O'Neill DW, Adams S, Bhardwaj N. Manipulating dendritic cell biology for the active immunotherapy of cancer. Blood. 2004;104:2235-2246. [60] Schettini J, Mukherjee P. Physiological Role of Plasmacytoid Dendritic Cells and Their Potential Use in Cancer Immunity. Clin Dev Immunol. 2008;2008:1-10. [61] Liu Y-J. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol. 2005;23:275-306. [62] McKenna K, Beignon A-s, Bhardwaj N. MINIREVIEW Plasmacytoid Dendritic Cells : Linking Innate and Adaptive Immunity. 2005;79:17-27. [63] Aarntzen EH, Figdor CG, Adema GJ, Punt CJ, de Vries IJ. Dendritic cell vaccination and immune monitoring. Cancer Immunol Immunother. 2008;57:1559-1568. [64] Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy. Ann Rev Med. 1999;50:507-529. [65] Sabatté J, Maggini J, Nahmod K, Amaral MM, Martínez D, Salamone G, et al. Interplay of pathogens, cytokines and other stress signals in the regulation of dendritic cell function. Cytokine Growth Factor Rev. 2007;18:5-17. [66] Riboldi E, Musso T, Moroni E, Urbinati C, Bernasconi S, Rusnati M, et al. Cutting Edge: Proangiogenic Properties of Alternatively Activated Dendritic Cells. J Immunol. 2005;175:2788-2792. [67] Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol. 2002;20:621-667. [68] Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Review Immunol. 2003;21:685-711. [69] de Jong EC, Smits HH, Kapsenberg ML. Dendritic cell-mediated T cell polarization. Springer Semin Immunopathol. 2005;26:289-307. [70] M F, Maini R. Discovery of TNF-alpha as a therapeutic target in rheumatoid arthritis: preclinical and clinical studies. Joint Bone Spine. 2002;69:12-18. [71] Benencia F, Sprague L, McGinty J, Pate M, Muccioli M. Dendritic cells the tumor microenvironment and the challenges for an effective antitumor vaccination. J Biomed Biotechnol. 2012;2012:425476-425476. [72] Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol. 2003;3:984-993. eng. [73] Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein Ra, Bazan F, et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med. 2001;194:863-869. [74] Kumar H, Kawai T, Akira S. Pathogen recognition in the innate immune response. Biochem J. 2009;420:1-16. [75] Ireton RC, Gale M. RIG-I like receptors in antiviral immunity and therapeutic applications. Viruses. 2011;3:906-919. [76] Janeway Ca, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216. [77] Knapp S. Update on the role of Toll-like receptors during bacterial infections and sepsis. Wiener medizinische Wochenschrift (1946). 2010;160:107-111. [78] Schreibelt G, Tel J, Sliepen KEWJ, Benitez-Ribas D, Figdor C, Adema G, et al. Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy. Cancer Immunol Immunother. 2010;59:1573-1582. [79] Diebold S. Activation of Dendritic Cells by Toll-Like Receptors and C-Type Lectins. In: Lombardi G, Riffo-Vasquez Y, editors. Dendritic Cells. Handbook of Experimental Pharmacology. 188: Springer Berlin Heidelberg; 2009. p. 3-30. [80] Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278:8869-8872. [81] Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, Crespo J, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J Biol Chem. 2003;278:5509-5512. [82] Shaw MH, Reimer T, Kim Y-g, Nuñez G. NIH Public Access. 2009;20:377-382. [83] Unger WWJ, van Kooyk Y. ‘Dressed for success’ C-type lectin receptors for the delivery of glyco-vaccines to dendritic cells. Curr Opin Immunol. 2011;23:131-137. [84] van Kooyk Y. C-type lectins on dendritic cells: key modulators for the induction of immune responses. Biochem Soc Trans. 2008;36:1478-1481. [85] Burgdorf S, Kautz A, Böhnert V, Knolle PA, Kurts C. Distinct Pathways of Antigen Uptake and Intracellular Routing in CD4 and CD8 T Cell Activation. Science. 2007;316:612-616. [86] Adams S, O'Neill DW, Bhardwaj N. Recent advances in dendritic cell biology. J Clin Immunol. 2005;25:87-98. [87] Kadowaki N. Dendritic cells: a conductor of T cell differentiation. Allergol Int. 2007;56:193-199. eng. [88] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu Y, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767-811. [89] AK P, H U, JS F, Banchereau J. Dendritic cells: a critical player in cancer therapy? J Immunother. 2008;31:793-805. [90] Hubbell JA, Thomas SN, Swartz MA. Materials engineering for immunomodulation. Nature. 2009;462:449-460. [91] Raghavan M, Del Cid N, Rizvi SM, Peters LR. MHC class I assembly: out and about. Trends Immunol. 2008;29:436-443.

© FORMATEX 2013

1701


[92] Kim J, Mooney DJ. In Vivo Modulation of Dendritic Cells by Engineered Materials: Towards New Cancer Vaccines. Nano today. 2011;6:466-477. [93] Hwang I, K D. Receptor-mediated T cell absorption of antigen presenting cell- derived molecules. Front Bioscience. 2011;16:411-421. [94] Kalinski P. Dendritic cells in immunotherapy of established cancer: Roles of signals 1, 2, 3 and 4. Curr Opin Invest Drugs. 2009;10:526-535. [95] Takeuchi S, Furue M. Dendritic cells-ontogeny. Allergol Int. 2007;56:215-223. [96] Fievez V, Plapied L, des Rieux A, Pourcelle V, Freichels H, Wascotte V, et al. Targeting nanoparticles to M cells with nonpeptidic ligands for oral vaccination. Eur J Pharm Biopharm. 2009;73:16-24. [97] Watts PJ, Smith A. Re-formulating drugs and vaccines for intranasal delivery: maximum benefits for minimum risks? Drug Discov Today. 2011;16:4-7. [98] Krauland AH, Guggi D, Bernkop-Schnurch A. Thiolated chitosan microparticles: a vehicle for nasal peptide drug delivery. Int J Pharm. 2006;307:270-277. [99] Alpar H, JE E, ED W, S S. Intranasal vaccination against plague, tetanus and diphtheria. Adv Drug Deliv Rev. 2001;51:173201. [100] Plotkin SA. Vaccines: the fourth century. Clin Vacc Immunol. 2009;16:1709-1719. [101] Verardi PH, Titong A, Hagen CJ. A vaccinia virus renaissance: new vaccine and immunotherapeutic uses after smallpox eradication. Hum Vaccines Immunother. 2012;8:961-970. [102] Evans TG, Brennan MJ, Barker L, Thole J. Preventive vaccines for tuberculosis. Vaccine. 2013;31 Suppl 2:B223-226. [103] Franca RF, da Silva CC, De Paula SO. Recent advances in molecular medicine techniques for the diagnosis, prevention, and control of infectious diseases. Eur J Clin Microbiol Infect Dis. 2013;32:723-728. [104] Abebe F, Bjune G. The protective role of antibody responses during Mycobacterium tuberculosis infection. Clin Exp Immunol. 2009;157:235-243. [105] Titball RW. Vaccines against intracellular bacterial pathogens. Drug Discov Today. 2008;13:596-600. [106] Schenkel JM, Fraser KA, Vezys V, Masopust D. Sensing and alarm function of resident memory CD8(+) T cells. Nature Immunol. 2013;14:509-513. [107] Igietseme J, Eko F, He Q, Black C. Antibody regulation of Tcell immunity-implications for vaccine strategies against intracellular pathogens. Expert Rev Vaccines. 2003;3:23-34. [108] Casadevall A, Pirofski L-a. Antibody-mediated regulation of cellular immunity and the inflammatory response. Trends Immunol. 2003;24:474-478. [109] McEwan WA, Tam JC, Watkinson RE, Bidgood SR, Mallery DL, James LC. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nature Immunol. 2013;14:327-336. [110] Kaur J, Jain SK. Role of antigens and virulence factors of Salmonella enterica serovar Typhi in its pathogenesis. Microbiol Res. 2012;167:199-210. [111] Camejo A, Carvalho F, Reis O, Leitão E, Sousa S, Cabanes D. The arsenal of virulence factors deployed by Listeria monocytogenes to promote its cell infection cycle. Virulence. 2011;2:379-394. [112] Barat S, Willer Y, Rizos K, Claudi B, Mazé A, Schemmer AK, et al. Immunity to Intracellular Salmonella Depends on Surface-associated Antigens. PLoS Pathog. 2012;8:e1002966. [113] Zepp F. Principles of vaccine design-Lessons from nature. Vaccine. 2010;28 Suppl 3:C14-24. [114] Sette A, Rappuoli R. Reverse vaccinology: developing vaccines in the era of genomics. Immunity. 2010;33:530-541. [115] Mueller S, Papamichail D, Coleman JR, Skiena S, Wimmer E. Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity. J Virol. 2006;80:96879696. [116] Coban C, Koyama S, Takeshita F, Akira S, Ishii K. Molecular and cellular mechanisms of DNA vaccines. Hum Vaccines. 2008;4:453-456. [117] Tobin GJ, Trujillo JD, Bushnell RV, Lin G, Chaudhuri AR, Long J, et al. Deceptive imprinting and immune refocusing in vaccine design. Vaccine. 2008;26:6189-6199. [118] CP L, Paidhungat M, Whalen R, Punnonen J. DNA shuffling and screening strategies for improving vaccine efficacy. DNA Cell Biol. 2005;24:256-263. [119] Loessner H, Weiss S. Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther. 2004;4:157168. [120] Vonka V. Comments on therapeutic cancer vaccines. Immunotherapy. 2012;4:133-135. [121] M P. Improving vaccine delivery using novel adjuvant systems. Hum Vaccines. 2008;4:262-270. [122] Malyala P, O'Hagan DT, Singh M. Enhancing the therapeutic efficacy of CpG oligonucleotides using biodegradable microparticles. Adv Drug Deliv Rev. 2009;61:218-225. [123] Schlosser E, Mueller M, Fischer S, Basta S, Busch DH, Gander B, et al. TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses. Vaccine. 2008 Mar 20;26:1626-1637. [124] Krishnamachari Y, Geary SM, Lemke CD, Salem AK. Nanoparticle delivery systems in cancer vaccines. Pharm Res. 2011;28:215-236. [125] Trombetta ES, Mellman I. Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol. 2005;23:975-1028.. [126] Norbury CC. Drinking a lot is good for dendritic cells. Immunology. 2006;117:443-451. [127] Yi X, Shi X, Gao H. Cellular Uptake of Elastic Nanoparticles. Phys Rev Lett. 2011;107. [128] Tzlil S, Deserno M, Gelbart WM, Ben-Shaul A. A statistical-thermodynamic model of viral budding. [129] Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nature Revs Immunol. 2010;10:787-796.

1702

© FORMATEX 2013


[130] D H, Margaritis A. Biopolymer nanoparticle production for controlled release of biopharmaceuticals. Crit Rev Biotechnol. 2013. [131] Ali M, Brocchini S. Synthetic approaches to uniform polymers. Adv Drug Deliv Revs. 2006;58:1671-1687. [132] Ulery BD, Nair LS, Laurencin CT. Biomedical Applications of Biodegradable Polymers. J Polym Sci Part B, Polymer physics. 2011;49:832-864. P [133] Pulapura S Fau - Kohn J, Kohn J. Trends in the development of bioresorbable polymers for medical applications. J Biomater Appl.1992;6:216-250. [134] Chye Joachim Loo S, Ooi CP, Hong Elyna Wee S, Chiang Freddy Boey Y. Effect of isothermal annealing on the hydrolytic degradation rate of poly(lactide-co-glycolide) (PLGA). Biomaterials. 2005;26:2827-2833. [135] Li S. Hydrolytic degradation characteristics of aliphatic polyesters derived from lactic and glycolic acids. J Biomed Mater Res.1999;342-353. [136] Göpferich A, Tessmar J. Polyanhydride degradation and erosion. Adv Drug Deliv Rev. 2002;54:911-931. [137] Ford Versypt AN, Pack DW, Braatz RD. Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres — A review. J Control Release. 2013;165:29-37. [138] Houchin ML, Topp EM. Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. Journal Pharm Sciences. 2008;97:2395-2404. [139] Seyednejad H, Ghassemi AH, van Nostrum CF, Vermonden T, Hennink WE. Functional aliphatic polyesters for biomedical and pharmaceutical applications. J Control Release. 2011 May 30;152:168-176. PubMed PMID: 21223989. [140] Athanasiou K, Niederauer G, Agrawal C. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acidpolyglycolic acid copolymers. Biomaterials. 1996;17:93-102. [141] Hyon S, Jamshidi K, Ikada Y. Synthesis of polylactides with different molecular weights. Biomaterials. 1997;18:1503-1508. [142] Albertsson A, Varma I. Recent developments in ring opening polymerization of lactones for biomedical applications. Biomacromolecules. 2003;4:1466-1486. [143] Thomas CM. Stereocontrolled ring-opening polymerization of cyclic esters: synthesis of new polyester microstructures. Chem Soc Rev. 2010;39:165-173. [144] Kobayashi S, Uyama H, Kimura S. Enzymatic Polymerization. Chem Rev. 2001;101:3793-3818. [145] Dordick JS. Enzymatic and chemoenzymatic approaches to polymer synthesis. Trends Biotechnol. 1992;10:287-290. [146] Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Preat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release. 2012;161:505-522. [147] Lasprilla AJ, Martinez GA, Lunelli BH, Jardini AL, Filho RM. Poly-lactic acid synthesis for application in biomedical devices - a review. Biotechnol Adv. 2012;30:321-328. [148] Gunatillake P, Mayadunne R, Adhikari R. Recent developments in biodegradable synthetic polymers. In: El-Gewely MR, editor. Biotechnol Annu Rev. Volume 12: Elsevier; 2006. p. 301-347. [149] Woodruff MA, Hutmacher DW. The return of a forgotten polymer—Polycaprolactone in the 21st century. Prog Polym Sci. 2010;35:1271-1256. [150] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices... Biomaterials. 2000;21:2475-2490. [151] Soderquist RG, Sloane EM, Loram LC, Harrison JA, Dengler EC, Johnson SM, et al. Release of plasmid DNA-encoding IL-10 from PLGA microparticles facilitates long-term reversal of neuropathic pain following a single intrathecal administration. Pharm Res. 2010;27:841-854.. [152] Cun D, Jensen DK, Maltesen MJ, Bunker M, Whiteside P, Scurr D, et al. High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization. Eur J Pharm Biopharm. 2011;77:26-35. [153] Solbrig C, Saucier-Sawyer J, Cody V, Saltzman W, Hanlon DJ. Polymer nanoparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells. Molr Pharm. 2006;4:47-57. [154] Clawson C, Huang CT, Futalan D, Seible DM, Saenz R, Larsson M, et al. Delivery of a peptide via poly(D,L-lactic-coglycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity. Nanomedicine. 2010;6:651-661. [155] Diwan M Fau - Elamanchili P, Elamanchili P Fau - Lane H, Lane H Fau - Gainer A, Gainer A Fau - Samuel J, Samuel J. Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses. J Drug Target. 2003;11:495-507. [156] Prasad S, Cody V, Saucier-Sawyer JK, Saltzman WM, Sasaki CT, Edelson RL, et al. Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy. Nanomedicine. 2011;7:1-10. [157] Thomas C, Rawat A, Hope-Weeks L, Ahsan F. Aerosolized PLA and PLGA nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis B vaccine. Mol Pharms. 2011;8:405-415. [158] Tian J, Yu J. Poly(lactic-co-glycolic acid) nanoparticles as candidate DNA vaccine carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus. Fish & shellfish immunology. 2011 Jan;30:109-117. [159] Jain RA, Rhodes CT, Railkar AM, Malick AW, Shah NH. Controlled release of drugs from injectable in situ formed biodegradable PLGA microspheres: effect of various formulation variables. Eur J Pharm Biopharm. 2000;50(2):257–62. [160] Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003;55:329-347. [161] Tacken PJ, Figdor CG. Targeted antigen delivery and activation of dendritic cells in vivo: steps towards cost effective vaccines. Semin Immunol. 2011 Feb;23:12-20. [162] Benoit M, Baras B, Gillard J. Preparation and characterization of protein-loaded poly(epsilon-caprolactone) microparticles for oral vaccine delivery. Int J Pharm. 1999;184:73-84. [163] Corrigan OI, Li X. Quantifying drug release from PLGA nanoparticulates. Eur J Pharm Sci. 2009;37:477-485. [164] Gao H, Wang YN, Fan YG, Ma JB. Conjugates of poly(DL-lactide-co-glycolide) on amino cyclodextrins and their nanoparticles as protein delivery system. J Biomed Mater Res A. 2007;80:111-122.

© FORMATEX 2013

1703


[165] Manish M, Rahi A, Kaur M, Bhatnagar R, Singh S. A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge. PloS one. 2013;8:e61885. [166] Hamdy S, Molavi O, Ma Z, Haddadi A, Alshamsan A, Gobti Z, et al. Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+ T cell-mediated anti-tumor immunity. Vaccine. 2008;26:50465057. [167] Gamvrellis A, Leong D, Hanley JC, Xiang SD, Mottram P, Plebanski M. Vaccines that facilitate antigen entry into dendritic cells. Immunol Cell Biol. 2004;82:506-516. [168] Sutmuller RP, den Brok MH, Kramer M, Bennink EJ, Toonen LW, Kullberg BJ, et al. Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest. 2006;116:485-494. [169] Yang Y, Huang CT, Huang X, Pardoll DM. Persistent Toll-like receptor signals are required for reversal of regulatory T cellmediated CD8 tolerance. Nature Immunol. 2004;5:508-515. [170] Park K. Mechanism of cross-presentation of microencapsulated antigen. J Control Release. 2011;151:219. [171] Panyam J, Zhou W, Prabha S, Sahoo SK, Labhasetwar V. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles-implications for drug and gene delivery. FASEB. 2002;16:1217-1226. [172] Mallapragada SK, Narasimhan B. Immunomodulatory biomaterials. Int J Pharm. 2008;364:265-271. [173] Storni T, Kundig TM, Senti G, Johansen P. Immunity in response to particulate antigen-delivery systems. Adv Drug Deliv Rev. 2005 Jan 10;57:333-355. [174] Moghimi SM, Andersen AJ, Ahmadvand D, Wibroe PP, Andresen TL, Hunter AC. Material properties in complement activation. Adv Drug Deliv Rev. 2011;63:1000-1007. [175] Mosqueira V, Legrand P, Gulik A, Bourdon O, Gref R, Labarre D, et al. Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules. Biomaterials. 2001;22:2967-2979. [176] Nicolete R, dos Santos DF, Faccioli LH. The uptake of PLGA micro or nanoparticles by macrophages provokes distinct in vitro inflammatory response. Intl Immunopharmacol. 2011;11:1557-1563. [177] Fischer S, Uetz-von Allmen E, Waeckerle-Men Y, Groettrup M, Merkle HP, Gander B. The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles. Biomaterials. 2007;28:9941004. [178] Zaman M, Good MF, Toth I. Nanovaccines and their mode of action. Methods. 2013.60;226-231. [179] Nguyen DN, Mahon KP, Chikh G, Kim P, Chung H, Vicari AP, et al. Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery. Proc Nat Acad Sci. 2012;109:E797-E803. [180] Hamdy S, Haddadi A, Hung RW, Lavasanifar A. Targeting dendritic cells with nano-particulate PLGA cancer vaccine formulations. AdvDrug Deliv REv 2011;63:943-955. [181] Hillaireau H, Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. Cellular and molecular life sciences : CMLS. 2009;66:2873-2896. [182] Ehrlich M, Boll W, Van Oijen A, Hariharan R, Chandran K, Nibert ML, Kirchhausen T. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell. 2004;118:591-605. [183] Wang Z Fau - Tiruppathi C, Tiruppathi C Fau - Minshall RD, Minshall Rd Fau - Malik AB, Malik AB. Size and dynamics of caveolae studied using nanoparticles in living endothelial cells. ACS Nano. 2009;3:4410-4116. [184] Owens DE, 3rd, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm. 2006;307:93-102. [185] Xiang SD, Scholzen A, Minigo G, David C, Apostolopoulos V, Mottram PL, et al. Pathogen recognition and development of particulate vaccines: does size matter? Methods. 2006;40:1-9. [186] Gan Q, Dai D, Yuan Y, Qian J, Sha S, Shi J, et al. Effect of size on the cellular endocytosis and controlled release of mesoporous silica nanoparticles for intracellular delivery. Biomed Microdevices. 2012;14:259-270. [187] Gao H, Shi W, Freund LB. Mechanics of receptor-mediated endocytosis. Proc Natl Acad Sci U.S.A. 2005;102:9469-9474. [188] IF C. Evidence based route of administration of vaccines. Human Vaccines. 2008;4:67-73. [189] Cone RA. Barrier properties of mucus. Adv Drug Deliv Rev. 2009;61:75-85. [190] Johansen P, Mohanan D, Martinez-Gomez JM, Kundig TM, Gander B. Lympho-geographical concepts in vaccine delivery. J Control Release. 2010;148:56-62. [191] Cho K, Wang X, Nie S, Chen ZG, Shin DM. Therapeutic nanoparticles for drug delivery in cancer. Clin Cancer Research. 2008;14:1310-1316. [192] Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38:1404-1413. [193] Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm. 2005;298:315-322. [194] Vasir JK, Labhasetwar V. Quantification of the force of nanoparticle-cell membrane interactions and its influence on intracellular trafficking of nanoparticles. Biomaterials. 2008;29:4244-4252. [195] Thurn KT, Brown E, Wu A, Vogt S, Lai B, Maser J, et al. Nanoparticles for applications in cellular imaging. Nanoscale Res Let. 2007;2:430-441. [196] Liu J, Bauer H, Callahan J, Kopeckova P, Pan H, Kopecek J. Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics. J Control Release. 2010;143:71-79. [197] Yue ZG, Wei W, Lv PP, Yue H, Wang LY, Su ZG, et al. Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles. Biomacromolecules. 2011;12:2440-2446. [198] Zhao F, Zhao Y, Liu Y, Chang X, Chen C, Zhao Y. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small. 2011;7:1322-1337. [199] Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Nat Acad Sci. 2008;105:14265-14270.

1704

© FORMATEX 2013


[200] Lynch I, Cedervall T, Lundqvist M, Cabaleiro-Lago C, Linse S, Dawson KA. The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century. Adv Colloid Interface Sci. 2007;134-135:167-174. [201] Chiu YL, Ho YC, Chen YM, Peng SF, Ke CJ, Chen KJ, et al. The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan. J Control Release. 2010;146:152-159. [202] Venkataraman S, Hedrick JL, Ong ZY, Yang C, Ee PL, Hammond PT, et al. The effects of polymeric nanostructure shape on drug delivery. Adv Drug Deliv Rev. 2011 Nov;63:1228-1246. [203] Heng BC, Zhao X, Tan EC, Khamis N, Assodani A, Xiong S, et al. Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles. Arch Toxicol. 2011;85:1517-1528. [204] Chithrani Bd Fau - Chan WCW, Chan WC. Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Lett.7:1542-1550. [205] Alexis F, Prodgen E, Molnar L, Farokhzad O. Factors affecting the clearance and biodistribution of polymeric nanoparticles. MolPpharmaceut. 2008;5. [206] Kelly C, Jefferies C, Cryan SA. Targeted liposomal drug delivery to monocytes and macrophages. J Drug Deliv;2011:727241. [207] Burgdorf S, Kurts C. Endocytosis mechanisms and the cell biology of antigen presentation. Curr Opin Immunol. 2008;20:8995. [208] Fernandez-Megia E, Novoa-Carballal R, Quinoá E, Riguera R. Conjugation of bioactive ligands to PEG-grafted chitosan at the distal end of PEG. Biomacromolecules. 2007;8:833-842. [209] van den Berg JH, Oosterhuis K, Hennink WE, Storm G, van der Aa LJ, Engbersen JFJ, et al. Shielding the cationic charge of nanoparticle-formulated dermal DNA vaccines is essential for antigen expression and immunogenicity. J Control Release. 2010;141:234-240. [210] Takeuchi O Fau - Akira S, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;140:805-820. [211] Kawai T, Akira S. TLR signaling. Semin Immunol. 2007;19:24-32. [212] Florindo HF, Pandit S, Gonçalves LMD, Alpar HO, Almeida AJ. Streptococcus equi antigens adsorbed onto surface modified poly-ε -caprolactone microspheres induce humoral and cellular specific immune responses. Vaccine. 2008;26:4168-4177. [213] Guo C, Gemeinhart RA. Understanding the adsorption mechanism of chitosan onto poly(lactide-co-glycolide) particles. Eur J Pharm Biopharm. 2008;70:597-604. [214] Roth-Walter F, Scholl I, Untersmayr E, Ellinger A, Boltz-Nitulescu G, Scheiner O, et al. Mucosal targeting of allergen-loaded microspheres by Aleuria aurantia lectin. Vaccine. 2005;23:2703-2710. [215] Florindo HF, Pandit S, Gonçalves LMD, Videira M, Alpar O, Almeida AJ. Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres. Biomaterials. 2009;30:5161-5169. [216] Anish C, Goswami DG, Kanchan V, Mathew S, Panda AK. The immunogenic characteristics associated with multivalent display of Vi polysaccharide antigen using biodegradable polymer particles. Biomaterials. 2012;33:6843-6857. [217] Da Costa Martins R, Gamazo C, Sanchez-Martinez M, Barberan M, Penuelas I, Irache JM. Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles. J Control Release. 2012;162:553-560. [218] Taha MA, Singh SR, Dennis VA. Biodegradable PLGA85/15 nanoparticles as a delivery vehicle for Chlamydia trachomatis recombinant MOMP-187 peptide. Nanotechnology. 2012;23:325101. [219] Loucq C. Vaccines today, vaccines tomorrow: a perspective. Clini Exp Vaccine Res. 2013;2:4-7. [220] Lewis K. Platforms for antibiotic discovery. Nat Rev Drug Discov 2013;12:371-387. [221] FDA. FDA Guidance documents for anti-microbial clinical development [cited 2013 5th June]. Available from: http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm064980.htm. [222] European Medicines Agency, Committee for Medicinal Products for Human Use (CHMP), Guideline on the evaluation of medicinal products indicated for treatment of bacterial infections. [cited 2013 5th June] Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003417.pdf. [223] Sathish JG, Sethu S, Bielsky MC, de Haan L, French NS, Govindappa K, et al. Challenges and approaches for the development of safer immunomodulatory biologics. Nat Rev Drug Discov. 2013;12:306-324. [224] Peter PW, Moghimi SM. Complement Sensing of Nanoparticles and Nanomedicines. Functional Nanoparticles for Bioanalysis, Nanomedicine, and Bioelectronic Devices Volume 2. ACS Symposium Series. 1113: American Chemical Society; 2012. p. 365-382. [225] McNeil SE. Nanoparticle therapeutics: a personal perspective. Wires Nanomed Nanobi. 2009;1:264-271. [226] Zolnik BS, González-Fernández Á, Sadrieh N, Dobrovolskaia MA. Minireview: Nanoparticles and the Immune System. Endocrinology. 2010;151:458-465.

© FORMATEX 2013

1705